The seeds industry can be split into two high-value, commercial sectors: seeds for field crops such as corn, oil seeds, sugar beet and cereals, and vegetable and flower seed. The scientific improvement of crop plants has gone through a succession of innovations leading to the development of hybrid varieties for many crops and, most recently, to the introduction of genetically enhanced crops. The worldwide commercial seeds market is valued at around $30 billion (International Seed Federation).
1. Importance of Seed Size
Yield in crop plants where seed is the harvested product is usually defined as weight of seed harvested per unit area (Duvick, 1992). Consequently, individual seed weight is regarded as a major determinant of yield. Increasing seed size is desirable because it may increase total yield (Reynolds et al., 2001). There is also evidence that seed size (weight) is positively correlated with a number of components of ‘seed quality’ such as the percentage of germination (Schaal, 1980; Alexander and Wulff, 1985; Guberac et al, 1998); time to emergence (Winn, 1985; Wulff, 1986); durability (survival under adverse growing conditions) (Krannitz et al, 1991; Manga and Yadav, 1995); and growth rate (Marshall, 1986). Seed quality is an important factor in the cost of production of commercial seed lots since these must be tested before sale. Consequently, increasing total seed weight, even without increases in total seed yield, may have economic benefits through improvements in seed quality. Conversely, decreasing seed size may also be desirable in some circumstances, for example by facilitating water uptake required for germination (Harper et al., 1970), or in plants grown for their fruit.
Modification of seed size is also likely to improve yield through increasing the ‘sink strength’ of the seed (i.e. its capacity to demand nutrients from the seed parent), or increasing the period in which the seed is acting as a strong sink. It is well established that the demands of sink organs such as seeds have significant control over the rate of photosynthesis and the movement of photoassimilates from source to sink tissues (Patrick and Offler, 1995; Paul and Foyer, 2001). In wheat, the seed parent can supply more nutrients than developing seeds are able to demand for the first 15-20 days after pollination (Austin, 1980). Therefore modifications that enable seeds to draw nutrients earlier in development, for example by speeding up seed growth, will allow seeds to capture resources that would otherwise be wasted. An ‘improved source-sink balance permitting higher sink demand during grainfilling’ has also been proposed as a method for increasing yield in wheat (Reynolds et al., 2001).
2. Composition of Seeds
Mature seeds of flowering plants consist of three components: the seed coat, which is of exclusively maternal origin; and the two fertilization products, embryo and endosperm, which have maternal and paternal genetic contributions. Seeds develop from fertilized ovules. Ovule development has been described for many species (Bouman, 1984), including Arabidopsis thaliana (Robinson-Beers et al., 1992; Schneitz et al., 1995). The main structures of the mature ovule are: the embryo sac, which contains the female reproductive cells (egg and central cell); the nucellus, which surrounds the embryo sac at least partially; and the inner and outer integuments, which envelop the embryo sac and nucellus. After fertilization the embryo and nutritive endosperm develop inside the embryo sac while the integuments differentiate into the seed coat, which expands to accommodate the growing endosperm and embryo.
Most monocotyledonous plants, e.g. cereals including maize, wheat, rice, and barley (see Esau, 1965), produce albuminous seeds—that is, at maturity they contain a small embryo and a relatively massive endosperm. Most dicotyledonous plants, e.g. Brassica napus, (oil seed rape, canola), soybean, peanut, Phaseolus vulgaris (e.g. kidney bean, white bean, black bean) Vicia faba (broad bean), Pisum sativum (green pea), Cicer arietinum (chick pea), and Lens culinaris (lentil), produce exalbuminous seeds—that is, the mature seeds lack an endosperm. In such seeds the embryo is large and generally fills most of the volume of the seed, and accounts for almost the entire weight of the seed. In exalbuminous seeds the endosperm is ephemeral in nature and reaches maturity when the embryo is small and highly immature (usually heart/torpedo stage). Commonly embryo development depends on the presence of the endosperm, which is generally accepted to act as a source of nutrition for the embryo.
3. Control of Seed Size
Seed size control can be viewed from the perspective of (1) ‘development’—the extent of cell division and expansion in one or more seed components (e.g. Reddy and Daynard, 1983; Swank et al, 1987; Scott et al., 1998; Garcia et al., 2003) or (2) ‘metabolism’—metabolic activity and transport of nutrients within the seed and between the seed and seed parent (e.g. Weber et al., 1996, 1997). Development and metabolism are interdependent: for example, invertase activity (involved in hexose transport) at the boundary of maternal tissues and endosperm or embryo sac is required for endosperm proliferation in maize (Cheng et al., 1996), and high invertase activity is correlated with increased cell numbers in broad bean seed coat (Weber et al., 1996), legume embryos (Weber et al., 1997), and barley endosperm (Weschke et al., 2003). Our present investigations focus on the developmental aspects of seed size control, although it can be assumed that changes to cell division/expansion in the seed will also be correlated with changes in metabolic activity and nutrient flow.
3a. Endosperm-LED Seed Growth
Several studies show a correlation between endosperm growth and final seed size, for example in maize (Lin, 1984; Jones et al., 1996), and even in the dicot Arabidopsis thaliana, which has an ephemeral endosperm (Scott et al., 1998; Garcia et al., 2003). Work in our laboratory has shown that overproliferation of the endosperm leads to large seeds with large embryos, while inhibition of endosperm proliferation produces small seeds with small embryos. We have manipulated endosperm proliferation and seed size using a variety of methods, including modifications to the ratio of paternally to maternally inherited chromosomes in the endosperm, cytosine methylation status of the parents contributing to the seed, and use of the fis3/fie mutation (Scott et al., 1998; Adams et al., 2000; Vinkenoog et al., 2000). In these experiments we considered the resultant changes to seed growth to be ‘endosperm-led’, and effects on the embryo and the seed coat to be indirect. Some of our experiments specifically ruled out a direct effect on seed coat growth because the seed parent was wild-type and only the fertilization products were directly modified: for example, in the case of wild-type diploid seed parents crossed with tetraploid pollen parents, which produce large seeds (Scott et al., 1998), or wild-type seed parents crossed with pollen parents hypomethylated by a DNA METHYLTRANSFERASE 1 antisense construct, which produce small seeds (Adams et al., 2000). Similarly, Garcia et al. (2003) described the haiku mutants of Arabidopsis thaliana, which produce small seeds due to early arrest of endosperm proliferation. The authors also noted a failure of cell elongation after fertilization in the integuments of haiku mutants, and concluded this was an indirect effect of limited endosperm growth.
3b Role of Integuments/Seed Coat in Establishing Seed Size
Alonso-Blanco et al. (1999) investigated seed size in wild-type plants of two Arabidopsis thaliana accessions, Cvi and Ler: seeds of the former weigh 80% more than seeds of the latter and are 20% longer. In both accessions, the authors found that ‘seed coat and endosperm growth preceded embryo growth, determining the overall final length of the embryo and the seed’. They did find that the outer layer of the mature seed coat has more cells in Cvi than Ler, but did not investigate or comment on whether these extra cells were formed before or after fertilization. Moreover, the authors' inspection of mature unfertilized ovules showed that ovules in Ler were slightly longer than in Cvi, and therefore the authors concluded that ‘ovule size differences could not account for the final Ler/Cvi seed size variation’. Their overall major conclusion was that ‘the larger size of Cvi seeds compared with Ler is mainly because of the faster and prolonged growth of the integuments and the endosperm’ (i.e. after fertilization); they did not address the question of whether this growth was led by the integuments or the endosperm. The authors suggested that the final cell number and size in the seed coat ‘may be determined during ovule development’, but significantly, there was no suggestion that a larger number of integument cells before fertilization was responsible for a larger final seed size.
Garcia et al. (2005) examined crosses between Arabidopsis mutant or transgenic plants that produce small seeds because of the inhibition of either endosperm growth or integument/seed coat growth. These authors proposed a model in which seed size is determined by a reciprocal interaction between endosperm growth and elongation of integument/seed coat cells. They also reported that genotypes with fewer cells in the integument compensate by increasing cell elongation. The authors concluded, ‘The final cell number in the integument [seed coat] is balanced by cell elongation and does not influence the size of the seed.’
Jofuku et al. (2005) and Ohto et al. (2005) reported that mutations in the APETALA2 (AP2) gene increase seed size; Jofuku et al. (2005) also found that suppression of AP2 activity through antisense or sense cosuppression had the same effect. ap2 mutant seeds have seed coat abnormalities including large and irregular outer integument cells, lack of mucilage, and hypersensitivity to bleach; and the increase in seed size was found to be a mainly (Jofuku et al., 2005) or wholly (Ohto et al., 2005) maternal effect. However, neither paper investigated any possible correlation between (1) seed size and (2) cell number or any other aspect of integument/seed coat morphology in ap2 mutants or transgenics.
Weber et al. (1996) compared growth of the seed coat in large- and small-seeded genotypes of Vicia faba (broad bean). They found that large-seeded genotypes contained more cells in the seed coat at 9 days after pollination, but cell numbers in the two genotypes were similar at 4 days after pollination. Therefore the number of cells in the integuments before fertilization could not be a factor in final seed size.
4. Relevant Patent Publications
(i) Fischer and Mizukami (2003), ‘Methods for Altering Organ Mass in Plants’, US Patent Application 20030159180
Mutations in the AINTEGUMENTA (ANT) gene of Arabidopsis thaliana prevent formation of the integuments (Klucher et al., 1996; Baker et al., 1997). Mizukami and Fischer (2000) describe the phenotype of Arabidopsis thaliana plants over-expressing the wild-type ANT gene under the control of the constitutive 35S promoter. Ectopic ANT expression increases the size of many plant organs including seeds, as well as causing male sterility through failure of anther dehiscence. Most of the transgenic plants are also female sterile ‘because of abnormally extended proliferation of the chalazal nucellar cells’. However weak overexpressers could generate seeds after hand-pollination with wild-type pollen. ‘The enlarged 35S::ANT fruit included T2 seeds that were larger than normal (not shown in the application), because of enlarged embryos.’ The size of unpollinated ovules, and the number or size of cells in the integuments/seed coat, were not investigated or discussed. The large seed size of 35S::ANT seeds was attributed only to size of the nucellus and embryo. US patent application no. 20030159180 describes uses of a modified ANT polypeptide for altering the size of plant organs including seeds. It was reported that the transgenic plants had varying degrees of fertility that were not correlated with organ size. There was no investigation of the effect of expressing the modified ANT polypeptide on integument or seed coat growth.
(ii) Jofuku and Okamuro (2001), ‘Methods for Improving Seeds’, U.S. Pat. No. 6,329,567
Mutations in the APETALA2 (AP2) gene increase seed size (Okamuro and Jofuku, 1997). The mutations have a maternal effect on seed size but the only phenotype described for the integument/seed coat in apt mutants is that the cells of the outer layer of the seed coat are enlarged with an irregular shape, along with some other morphological abnormalities (Jofuku et al., 1994). U.S. Pat. No. 6,329,567 describes methods of modulating seed mass using AP2 transgenes, but this patent does not assess any effect of the transgenes on the integuments or seed coat.
(iii) Lepiniec et al. (2003), ‘Regulating Nucleic Acid for Expressing a Polynucleotide of Interest Specifically in the Endothelium of a Plant Seed and Uses Thereof’, WO 03/012106 A2
The BANYULS (BAN) gene is expressed exclusively in the inner layer of the inner integument (this layer is also called the endothelium) in early seed development (pre-globular stage) (Devic et al., 1999). International patent application WO 03/012106 A2 describes use of the BAN promoter to drive expression of various genes specifically in the testa (the seed coat layer derived from the inner integument). The authors propose uses such as modifying the tannin or fibre composition, or the hormonal equilibrium, but no relevant expression cassettes were reported or described. Modification of seed size is also proposed but only in the context of reducing or ablating seeds in fruit crops. A BAN promoter::BARNASE construct was shown to ablate the endothelium.
(iv) Zinselmeier et al. (2000), ‘Regulated Expression of Genes in Plant Seeds’, WO00/63401
This patent application relates to expression of genes such as ipt that ‘affect metabolically effective levels of cytokinins in plant seeds, as well as in the maternal tissue from which such seeds arise, including developing ears, female inflorescences, ovaries, female florets, aleurone, pedicel, and pedicel-forming regions’, and to transgenic plants with enhanced levels of cytokinin that exhibit ‘improved seed size, decreased tip kernel abortion, increased seed set during unfavorable environmental conditions, and stability of yield’. A nucellus promoter (nucellus is the maternal tissue surrounding the embryo sac and enclosed within the integuments) is among those suggested for driving expression cassettes, but integuments are not specifically mentioned in the patent application, nor were any maternal tissue-specific expression cassettes described. The disclosure of this patent application is particularly concerned with maize.
(v) Scott (2002), ‘Modified Plants’, WO/0109299
This patent application relates to methods for controlling endosperm size and development through use of an antisense DNA METHYLTRANSFERASE 1 gene that reduces cytosine methylation. As described in WO01/09299, and in Section 3a, above, modification to the cytosine methylation status of the seed or pollen parent alters seed size by altering the rate and extent of endosperm proliferation. Therefore the disclosure of this patent application relates exclusively to ‘endosperm-led’ seed growth.
In summary, documents in the prior art do not include an understanding that altering the size of integuments specifically through increasing the number of cells before fertilization could affect seed size after fertilization. A small number of published papers and patent applications touch on a possible relationship between seed coat size and seed size but do not make a link between (1) integument growth pre-fertilization and (2) final seed size.
5. Integument-LED Seed Growth
We were surprised therefore to discover in our laboratory a mutant, termed the mnt-1 mutant, that produces enlarged seeds through a primary effect on the integuments. Specifically, we observed that the seed cavity (i.e. the space within the post-fertilization embryo sac) is longer than normal giving the embryo more space to grow as a result of an increase in cell number in the integument. This was particularly surprising in view of the earlier research mentioned in section 3a above which indicated that changes in seed growth were ‘endosperm-led’. It was also surprising in view of the work of Alonso-Blanco et al (1999) mentioned in section 3b above which did not suggest that an increase in number of integuments cells led to an increase in seed size; and also in view of the work of Weber et al 1999 who found similar numbers of cells in the seed coat in small and large-seeded genotypes of broad bean soon after fertilization; and also in view of the work of Garcia et al. (2005) who claimed that ‘The final cell number in the integument [seed coat] is balanced by cell elongation and does not influence the size of the seed’; and also in view of the work of Jofuku et al. (2005) and Ohto et al. (2005) who found a maternal effect of the ap2 mutation on seed size but did not report a correlation between cell number in the integuments or in the seed coat and final seed size.
6. Increased Stem Diameter in mnt-1 Mutants
Increased stem diameter is also desirable in agriculture, as it may lead to an increase in plant biomass, which may in turn increase yield (Reynolds et al., 2001). Increased stem diameter and biomass are also desirable in certain crops such as trees and vegetables. Thicker stems are also desirable because this trait increases resistance to lodging, a serious problem that reduces yields in crops including cereals (Zuber et al., 1999), soybean (Board, 2001), and oilseed rape (Miliuviene et al., 2004). A further aspect of the mnt-1 mutant phenotype is increased diameter of the stems.